The kinetochore is the site of force generation for chromosome motion during mitosis (cell division). During this process each individual kinetochore, of which there are two on a replicated chromosome, switches between no motion, motion toward the spindle pole to which it is attached (P motion), and motion away from that spindle pole (AP motion). During this time the plus ends of kinetochore microtubules (kMTs) terminate at the kinetochore outer plate, while the minus ends terminate near the spindle pole. This means that kMTs elongate during AP motion and shrink during P motion, with current evidence pointing to the kinetochore as the major site of addition or dissociation of microtubule subunits. Thus changes between growth and shrinkage of kMTs, which are presumably different states of kMT dynamic instability, must be coordinated with changes in the direction of motion. One current hypothesis (Mitchison, T.J., Ann. Rev. Cell Biol., 4(1988)527) is that during AP motion kMTs grow through the kinetochore outer plate to reach an internally located plus-end directed MT motor, while during P motion the ends of the shrinking kMTs terminated on the distal surface of the outer plate. We are testing this hypothesis by same-cell correlative LM/3D EM methods, developed over the past year, which allow us to compute tomographic 3D reconstructions of PtK1 kinetochores in cells that have been followed via video light microscopy up to the moment of fixation. The location of kMT ends in the tomographic reconstructions is then determined using a quantitative measurement scheme we have developed. Initial results from two kinetochores undergoing AP motion chromosomes show that the kMTs terminate on the distal surface of the outer plate without significant penetration. In contrast, on one metaphase kinetochore, and one kinetochore undergoing P motion, all kMTs measured show significant penetration with at least half going all the way through the outer plate. These preliminary results contradict the above hypothesis and imply that, at least during congression, kMTs are following the motion dictated by the kinetochore rather than initiating the direction of motion through changes in dynamic instability state. We are currently expanding the data set and developing methods to selectively stain the kMTs and outer plate to refine the assay. (This work has been abstracted to the 1995 ASCB meeting).